Seminal studies by Yamanaka and colleagues revealed that ectopic expression of certain transcriptional factors could induce pluripotency in somatic cells. These induced pluripotent stem cells (iPSC) self-renew and differentiate into a wide variety of cell types, making them an appealing option for disease- and patient-specific regenerative medicine therapies. They have been used to successfully model human disease and have great potential for use in drug screening and patient-specific cell therapy. Furthermore, iPSCs generated from diseased cells can serve as useful tools for studying disease mechanisms and potential therapies. However, much remains to be understood about the underlying mechanisms of reprogramming of somatic cells to iPSCs, and there is concern regarding potential clinical applications in the absence of mechanistic insights.
The set of factors (RFs) for reprogramming to pluripotency include Oct3/4, Sox2, c-Myc, Klf4, Lin28, and Nanog. Oct3/4 and Sox2 are transcription factors that maintain pluripotency in embryonic stem (ES) cells while Klf4 and c-Myc are transcription factors thought to boost iPSC generation efficiency. The transcription factor c-Myc is believed to modify chromatin structure to allow Oct3/4 and Sox2 to more efficiently access genes necessary for reprogramming while Klf4 enhances the activation of certain genes by Oct3/4 and Sox2. Nanog, like Oct3/4 and Sox2, is a transcription factor that maintains pluripotency in ES cells while Lin28 is an mRNA-binding protein thought to influence the translation or stability of specific mRNAs during differentiation. Recently, it has been shown that retroviral expression of Oct3/4 and Sox2, together with co-administration of valproic acid, a chromatin destabilizer and histone deacetylase inhibitor, is sufficient to reprogram fibroblasts into iPSCs.
To generate iPSCs from somatic cells, viral vectors or plasmids have been used to overexpress some combination of these reprogramming factors. However, these methods result in a low efficiency of reprogramming and fail to provide precise control of the reprogramming process. Furthermore, these methods for nuclear reprogramming inherently raise concerns about potential tumorigenicity and gene-silencing mutations caused by DNA integration. The integration of foreign DNA into the host genome from retroviral infection raises concerns that the integration of foreign DNA could silence indispensable genes or induce dysregulation of these genes. While Cre-LoxP site gene delivery or PiggyBac transposon approaches have been used to excise foreign DNA from the host genome following gene delivery, neither strategy eliminates the risk of mutagenesis because they leave a small insert of residual foreign DNA.
Recently, another form of nuclear reprogramming has involved the transdifferentiation of one somatic cell to another, using a similar approach as in generating iPSCs. However, in this case, most investigators have used exogenous DNA encoding lineage specific transcription factors. One can also employ conditional expression of the Yamanaka factors to induce “partially reprogrammed” cells, and then maintain the cells in medium that favors the generation of the somatic cell of interest. See Ginsberg et al. (2012) Cell 151(3):559-75; and Li et al. (2013) Arterioscler Thromb Vasc Biol. 33(6):1366-75.
Another form of nuclear reprogramming is related to telomere extension. Generally, somatic cells do not have telomerase activity. One can employ an activator of innate immunity and the telomerase enzyme and/or its RNA subunit TERC, to increase telomere length. In this case, the activator of innate immunity enhances the telomere extension as it increases endogenous telomerase activity and/or increases epigenetic plasticity so that the telomerase enzyme can interact more efficiently with the telomeric region of the chromosomes.
As an alternative to reprogramming with foreign DNA, approaches have been developed that transfer mRNA encoding reprogramming factors into somatic cells. A mmRNA based approach for iPSC generation or transdifferentiation to a different somatic cell type avoids concerns for integration of foreign DNA, and provides for greater control over the concentration, timing, and sequence of factor stimulation. Cytosolic delivery of mRNA into mammalian cells can be achieved using electroporation or by complexing the RNA with a cationic vehicle to facilitate uptake by endocytosis, or by incorporating the RNA in exosomes, nanoparticles or microparticles. See, for example Warren et al. (2010) Cell Stem Cell 7(5):618-630, review by Mandal and Rossi (2013) Nat. Protocol. 8(3):568-582; and Yakubov et al. (2010) Biochem Biophys Res Commun. 394(1):189-93. To enhance the functionality activity of the mRNA, synthetic messenger RNAs have incorporated modifications designed to bypass innate immune responses. Using In Vitro Co-Transcription capping reactions, synthetic mRNA for these purposes typically incorporates a chemically synthesized 5′ guanine cap which is a structural homolog of the natural 7-methylguanosine [m7G(5′)] cap. This commonly used type of in vitro Transcription (IVT) reaction, however, yields a significant fraction of uncapped RNA products primed with 5′ guanine triphosphates which resembles some viral and bacterial RNAs (see Lippincott Williams & Wilkins, Philadelphia, Pa., ed. 5, 2007; and Bieger et al. (1989) J. Bacteriol. 171:141. These RNA products can induce innate immune signaling as they are detected sensors for 5′ triphosphate ssRNA, see Fields Virology, D. Knipe, P. M. Howley, Eds.
Prior art approaches have sought to reduce the immunogenic profile of synthetic RNA by treatment with phosphatase in order to reduce activation of antiviral and bacterial defenses by single stranded 5′ triphosphate RNA. Additional modifications to reduce innate immune responses to transfected RNA can include the incorporation of modified ribonucleoside bases, for example 5-methylcytidine (5 mC) for cytidine, and/or pseudouridine (psi) for uridine. The cells can also be grown in media supplemented with a type I interferon decoy receptor, for example the vaccinia B18R protein.
However significant problems have remained in the actual practice of such methods. The present invention addresses this issue.